Phenotypic divergence in an island bee population: Applying geometric morphometrics to discriminate population‐level variation in wing venation

Abstract Phenotypic divergence is an important consequence of restricted gene flow in insular populations. This divergence can be challenging to detect when it occurs through subtle shifts in morphological traits, particularly in traits with complex geometries, like insect wing venation. Here, we employed geometric morphometrics to assess the extent of variation in wing venation patterns across reproductively isolated populations of the social sweat bee, Halictus tripartitus. We examined wing morphology of specimens sampled from a reproductively isolated population of H. tripartitus on Santa Cruz Island (Channel Islands, Southern California). Our analysis revealed significant differentiation in wing venation in this island population relative to conspecific mainland populations. We additionally found that this population‐level variation was less pronounced than the species‐level variation in wing venation among three sympatric congeners native to the region, Halictus tripartitus, Halictus ligatus, and Halictus farinosus. Together, these results provide evidence for subtle phenotypic divergence in an island bee population. More broadly, these results emphasize the utility and potential of wing morphometrics for large‐scale assessment of insect population structure.

Beyond these taxonomic biases, our understanding of phenotypic variation across populations is biased toward traits that are easily distinguished or quantified by human observers, such as body size and coloration (Doucet et al., 2004;Kraemer et al., 2019;Lomolino, 1985;Palkovacs, 2003). In contrast, variation in traits that present measurement challenges, such as morphological traits with complex geometries, tends to be underexplored. One such trait is the pattern of venation in insect wings. Veins provide the primary structural support for wings, and while the functional significance of variation in venation patterns remains largely unclear (Combes & Daniel, 2003), they are highly conserved in insect lineages and thus are useful in phylogenetic reconstructions and taxonomic determinations (Comstock & Needham, 1898;Sharkey & Roy, 2002). Indeed, many identifying characteristics in bee taxonomy are found in wing venation patterns, with characteristic variation distinguishing genera and species (Michener, 1994).
Within a species, however, wing venation may present subtler patterns of variation that are undetectable via traditional observation methods. Geometric morphometrics, a set of methods that allows for spatial analysis of biological forms, has emerged as a promising approach to quantifying variation in complex morphological traits (Mitteroecker & Gunz, 2009;Rohlf & Marcus, 1993). This approach has been successfully implemented to discriminate patterns of insect wing venation among (Baylac et al., 2003;Deregnaucourt et al., 2021;Francoy et al., 2009Francoy et al., , 2012Kaba et al., 2017;Perrard et al., 2014;Rattanawannee et al., 2010Rattanawannee et al., , 2015Santoso et al., 2018;Villemant et al., 2007) and even within species (Francisco et al., 2008;Francoy et al., 2011Francoy et al., , 2016. Geometric morphometrics, therefore, has potential to assess the extent of phenotypic divergence among discrete insect populations by quantifying variation in this highly conserved trait. We examined trait variation among island and mainland native bee populations in a Southern California coastal ecoregion. Santa Cruz Island is a 249 km 2 Pacific island located 32 km due south of mainland Santa Barbara, California. It is the largest of the California Channel Islands, an eight-island archipelago notable for its biodiversity and endemic species and which has served as a site for many microevolutionary studies of island-mainland variation (O'Reilly & Horn, 2004). Santa Cruz Island shares many of its bee fauna with mainland Santa Barbara (Seltmann, 2019), but the distance separating these locations generally precludes gene flow between populations. Bees typically forage within a few kilometers of their nesting sites, and dispersal distances are generally well under the 30 km water barrier separating Santa Cruz Island from the mainland (O'Reilly & Horn, 2004). Further, while stem-and wood-nesting bees have heightened island dispersal capabilities due to human transport of wood materials (Poulsen & Rasmussen, 2020), ground-nesting bees have limited opportunities for human-mediated island dispersal. Honey bees (Apis mellifera Linnaeus, 1758) were eradicated from the island by 2004 and have not been observed there since (Naughton et al., 2014;Seltmann et al., 2019;Wenner et al., 2009), suggesting that the channel is not easily crossed even by mediumsized bees. As such, we are confident that gene flow between island and mainland bee populations in this context is minimal to nonexistent, increasing the likelihood of phenotypic divergence between populations.
In this study, we investigate variation in wing venation in island and mainland populations of the sweat bee, Halictus tripartitus Cockerell, 1985. H. tripartitus is a widespread, ground-nesting social bee native to western North America and locally abundant both in mainland Santa Barbara and on Santa Cruz Island. We analyze museum specimens using a geometric morphometrics framework to assess the extent of variation in wing venation patterns between these two reproductively isolated populations. To contextualize the degree of variation, we additionally characterize variation in wing venation between H. tripartitus and two sympatric congeners, H. ligatus Say, 1837 and H. farinosus Smith, 1853. In doing so, we assess the role of reproductive isolation on population differentiation of morphological traits.

| Specimens and wing imaging
To assess population-level variation in wing venation patterns, we imaged wings from three Halictus species: H. tripartitus (n island = 149; n mainland = 149), H. ligatus (n mainland = 43), and H. farinosus (n island = 3; n mainland = 40; Figure 1). To achieve even sampling across species, we randomly selected 43 specimens of each species to analyze in our species-level comparison. We obtained bee specimens from the University of California, Santa Barbara Invertebrate Zoology Collection housed by the Cheadle Center for Biodiversity and Ecological Restoration. All specimens were female and were col-

| Data analysis
We Procrustes-aligned landmark coordinates using R package "geomorph" version 4.0.0 (Adams et al., 2022;Baken et al., 2021). To test for statistical differences between the two H. tripartitus populations and among the three species, we ran one-way multivariate analysis of variance (MANOVA) tests using R package "RRPP" version 1.3.1 (Collyer & Adams, 2018. To visualize separation among groups, we generated density plots with discriminant analysis of principal components (DAPC) using R package "adegenet" version 2.1.10 (Jombart, 2008;Jombart & Ahmed, 2011). To test the accuracy of using wing landmarks to predict an unknown bee's species or population, we utilized DAPC cross-validation. Cross-validation also informed the number of principal components (PCs) retained in each analysis (Jombart & Collins, 2015).

| RE SULTS
Our analysis of wing landmark coordinates successfully discriminated between wings of H. tripartitus, H. ligatus, and H. farinosus (MANOVA: Pillai = 1.817, p < .001); (full MANOVA tables in Table 1; landmark coordinates in Table S1). Based on cross-validation, 6 PCs were retained, and the density plot shows separation between species ( Figure 4a). The cross-validation test assigned 100% of Halictus specimens to their correct species ( Figure S1a

| DISCUSS ION
We employed a geometric morphometrics framework to demonstrate strong differentiation in wing venation geometries across three sympatric species of Halictus bees, as well as significant (though less pronounced) differentiation across island and mainland populations of H. tripartitus. Species-level variation in wing venation is well established for many bee taxa, serving as useful characters for identification to family, genus, or species levels (Michener, 1994).
Several studies have even successfully discriminated between distinct insect subspecies or genetic lineages within species using wing morphometrics (Akahira & Sakagami, 1959;Carneiro et al., 2019;Francoy et al., 2008Francoy et al., , 2011. Fewer studies, however, have investigated population-level variation in wing venation (Francoy et al., 2011;Rossa et al., 2016). This variation in wing morphology can serve as a useful proxy for assessing the extent of divergence between closely related populations (Oleksa & Tofilski, 2015;Peil & Aranda, 2021). More broadly, these results provide robust evidence for microevolutionary change in wing morphology across reproductively isolated bee populations.
As for many island bee populations, it is unknown how and when this population of H. tripartitus colonized Santa Cruz Island.
Island dispersal by bees is generally poorly understood, though phylogenetic analyses and behavioral studies can offer clues to potential avenues for colonization events. On Santa Cruz Island, colonization by many bee species could have occurred during the Last Glacial Maximum (17,000-18,000 years ago), when lowered sea levels reduced the distance from the mainland to about 6 km F I G U R E 1 Lateral images of female Halictus tripartitus (top; UCSB-IZC00040597), H. ligatus (middle; UCSB-IZC00044094), and H. farinosus (bottom; UCSB-IZC00042935) specimens. Images produced by Luz Ceja. (Miller, 1985). Gene flow across the channel may have continued for an unknown period, depending primarily on the distance of the water barrier and the dispersal capabilities of H. tripartitus.
Dispersal timing aside, it is evident from our results that the Santa observation via simple measurement techniques. The application of geometric morphometrics to insect wing venation patterns is still a relatively recent development, but already has shown promise for species identifications (Aytekin et al., 2007;Francoy et al., 2009;Rattanawannee et al., 2010). Our accurate discrimination between three Halictus species likewise supports a role for geometric morphometrics in taxonomic identification to the species level. Further, geometric wing morphometrics may be useful for distinguishing among populations (Henriques et al., 2020;Rossa et al., 2016) and between species within complexes (Francoy et al., 2011). Identifying features of wing venation have even been successfully integrated into computer-aided identification systems, which can accurately identify bee specimens to species and even subspecies from images of wings (Buschbacher et al., 2020;Rattanawannee et al., 2012).
Our results indicate that population variation in wing venation can be successfully discriminated using geometric morphometrics, and suggest that these patterns could be usefully extended toward automated identification systems with the aim of further classifying specimens to the population level.
Beyond its use in population identification, wing morphometry holds valuable potential for large-scale population studies, by providing a tractable alternative to more costly and time-consuming molecular methods for analyzing population structure. Unlike some morphological traits that can degrade over time, wing venation is strongly preserved in museum specimens, presenting opportunities for sampling of existing specimens in place of conducting new surveys. Wings represent powerful candidates for geometric morphometric analysis because their two-dimensional surfaces lend themselves to straightforward imaging, in contrast to threedimensional traits that require additional protocols to standardize orientation within images. Future studies seeking to identify bees to species or population level may find this methodology viable and potentially more adaptable than traditional taxonomic identifications using dichotomous keys. In particular, we envision that wing morphometrics could increase the feasibility of large-scale monitoring projects by reducing taxonomic labor (Engel et al., 2021).
In conclusion, we demonstrated species-and population-level variation in Halictus wing venation. Our results provide evidence for the divergence of wing venation patterns in isolated island and mainland populations of H. tripartitus. Our study emphasizes that wing venation patterns can act as quantifiable indicators of phenotypic differentiation within species, and may be useful for inferring the extent of variation among reproductively isolated populations.

ACK N OWLED G M ENTS
We would like to thank Zoe Wood, Evan Hobson, and Charles Braman for helpful discussion and comments on the manuscript, Dr. Christopher Evelyn for statistical guidance, UCSB Statistics Department for consultation, and Yolanda Diao for landmarking specimens. We also thank Luz Ceja for producing the lateral specimen images. We also thank reviewers for comments that improved the manuscript. Finally, we thank Jaime Pawelek for identification of bee specimens. This research was funded by a National Science

DATA AVA I L A B I L I T Y S TAT E M E N T
All data associated with this study are publicly available in Zenodo.